ANDROLOGY. Effects of Hydrogen Peroxide on Human Spermatozoa* ANDROLOGY. SERGIO OEHNINGER, 1'3 PETER B LACKMORE, ~ MARY MAHONY, ~ and GARY HODGEN 1

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1 ANDROLOGY ANDROLOGY Effects of Hydrogen Peroxide on Human Spermatozoa* SERGIO OEHNINGER, 1'3 PETER B LACKMORE, ~ MARY MAHONY, ~ and GARY HODGEN 1 Submitted: December 15, 1994 Accepted: January 4, 1995 Purpose: Reactive oxygen species (ROS) have been reported widely to cause deleterious effects on sperm viability and function due to peroxidation of membrane lipids. However, their action appears more selective at low concentrations; recent evidence indicates that the superoxide anion can promote capacitation and induce hyperactivated motility (HA) in human spermatozoa and that hydrogen peroxide (11202) may participate in capacitation of hamster spermatozoa. The objective of these studies was to investigate the direct effects of H202 on functions crucial to fertilization in human spermatozoa. Methods: In these prospective studies, we examined the dose- and time-dependent effects of H202 on sperm membrane-mediated events (binding to the zona pellucida and changes in intraceltular calcium concentration [Ca 2+]i, motility patterns, and acrosome reaction). Sperm from fertile donors were used in the experiments under capacitating conditions after separation of the motile fraction by wash~swim-up. [Ca2+] i was measured by the fluorescent fura-2 indicator, and sperm-zona pellucida binding was assessed with the hemizona assay (HZA). Hyperactivated motility was evaluated by computerized analysis, and the percentage of acrosome reacted sperm was detected by FITC-Pisum sativum lectin and indirect immuno fluorescence. Results: In the HZA, H20 z did not influence sperm-zona pellucida binding at low concentrations (0.05 mm and O. l mm), but significantly reduced binding at 0.2 mm (P < * Presented at the 50th Annual Meeting of the American Fertility Society, San Antonio, Texas, November 5-10, The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, 601 Colley Avenue, Norfolk, Virginia Department of Pharmacology, Eastern Virginia Medical School, Norfolk, Virginia To whom correspondence should be addressed vs controls) significantly decreased HA in a dose-dependent manner (P < ) and had a significant effect (P < 0.01) on acrosome reaction (stimulatory effect at 0.01 mm). H202 did not affect basal [Ca2+]i; however, He02 (0.1 mm through 10 mm) decreased the initial phase of progesterone-induced (P4:1 txm) enhancement of [Ca2+] i in a dose- and time-dependent fashion. Preincubation of sperm with catalase (20 txg/ml) potentiated the P4-induced increase of[ca 2 +]i did not significantly modify [Ca 2 +]i increase in response to inomycin (10 IxM). Conclusions: These experiments show that directly affects sperm Junctions crucial to fertilization in a doseand time-dependent fashion. Low concentrations maintain capacitation, whereas higher concentrations have deleterious effects, as determined by the end points of the capacitation process. The latter effects are probably dependent on modifications of plasma membrane and intracellular homeostasis by the oxidative process. KEY WORDS: hydrogen peroxide; reactive oxygen species; human sperm. INTRODUCTION Reactive oxygen species (ROS) have been widely reported to cause deleterious effects on sperm viability and function due to peroxidation of membrane lipids. Reactive oxygen species such as the superoxide anion, hydrogen peroxide (H202), and the hydroxyl radical can be produced by spermatozoa (1,2). Additionally, phagocytic leukocytes present in semen have also been shown to produce ROS (3,4). Due to their high content of polyunsaturated fatty acids, human spermatozoa are especially sensitive to ROS-induced damage and H202 appears to be the most toxic ROS under in vitro conditions (5-9). Investigations have shown that damaged or /95/ / Plenum Publishing Corporation

2 42 OEHNINGER~ BLACKMORE, MAHONY, AND HODGEN defective spermatozoa produce high levels of ROS, whereas sperm from fertile men (donors) do not (1,2,6,9). To counteract the effects of ROS, spermatozoa and seminal plasma possess systems to scavenge ROS and prevent intracellular damage, including the presence of superoxide dismutase, the glutathione peroxidase/reductase system, catalase, proteins (such as albumin) and other molecules (vitamin E and C, taurine, and hypotaurine) (5,8,10-13). Recently, it was demonstrated that the superoxide anion, generated by the combination of xanthine plus xanthine oxidase (in the presence of catalase), induced hyperactivated motility (HA) and capacitation in human spermatozoa (14). Furthermore, H202 was also shown to be involved in the capacitation process of hamster spermatozoa (15). Because of the dual effects observed with H202 (deleterious effects at high concentrations, and more selective, positive effects on capacitation at low concentrations), we designed these studies to examine dose- and time-dependent effects of H202 on functions crucial to fertilization in human spermatozoa. These functions included sperm-zona pellucida binding, HA, acrosome reaction and its prerequisite, an increase in the intraceuular levels of calcium [Ca2+]i. MATERIALS AND METHODS Semen Donors and Sperm Preparation Semen samples were obtained from four normozoospermic men (donors) by masturbation after 2 to 4 days of sexual abstinence. These men were active participants of the donor insemination program of our Institute, had fathered a pregnancy within the last year, and had negative bacteriologic semen cultures. All semen samples were studied after liquefaction was complete (<30 min) and within 1 h of collection. Following liquefaction, the semen samples were subjected to a basic evaluation of sperm parameters (concentration, progressive motility, and morphology as previously published) followed by a swim-up separation of the sperm motile fraction (16). All ejaculates used in the experiments had an original sperm concentration t>60 x 106/ml, progressive motility >~50%, normal morphology >14% (strict criteria), and < /ml leukocytes (by peroxidase staining). For the swim-up separation, 1 ml of semen was diluted with I ml of medium con- sisting of Ham's F-10 (Gibco Lab., Grand Island, NY) supplemented with 0.3% human serum albumin (HSA) (Irvine Sci., Santa Anna, CA) and centrifuged gently at 290 g for 8 min. The sperm pellet was washed by centrifugation for 5 rain. Finally, 1 ml of medium was gently layered over the undisturbed pellets, and specimens were incubated for 1 h at 37~ in 5% CO2 in air. The supernatant was removed and adjusted to appropriate concentrations depending on experiments. Hyperactivated Motility Motility characteristics were objectively measured with the HTM-2030 motility analyzer (Hamilton-Thorn Research, Danvers, MA) using fixed parameter settings previously established (17). Each sample (a 5-1xl aliquot of swim-up adjusted to 20 x 10 6 motile sperm/ml) was loaded by capillary action into a microcell counting chamber (20 p~m deep) and the tube transferred to the HTM-2030 where it was maintained at 37~ for 1 min prior to the start of data acquisition. Data collection was completed on randomly selected fields until at least 100 motile spermatozoa were analyzed. Motility patterns consistent with HA were identified as previously described (17,18). Hemizona Assay Tight binding of sperm to the human zona pellucida was assessed using the HZA as previously detailed (19,20). Briefly, salt-stored human oocytes were microbisected into matching hemizonae using Narishige micromanipulators (Narishige, Tokyo, Japan) mounted on a phase-contrast inverted microscope (Nikon Diaphot, Garden City, NY). Control and test sperm droplets (100 txl of a final dilution of 0.5 x 105 motile sperm/ml) were separately incubated under oil with the matching hemizonae for 4 h at 37~ in 5% CO2 in air. After the coincubation period, the hemizonae were washed to remove loosely attached sperm using a finely drawn glass pipette and the number of sperm tightly bound to the outer zona surface was counted under phasecontrast microscopy ( 200). Acrosomal Status The proportion of acrosome-reacted sperm was determined using the fluorescent probe fluorescein isothiocyanate (FITC)-labeled Pisum sativum lectin

3 DUAL EFFECTS OF H202 ON HUMAN SPERM FUNCTION 43 (PSA), following previously described methods (22). Briefly, 20 Ixl of final sperm suspensions at the concentration of 0.5 x 105 motile sperm/ml were smeared on microscope slides and air-dried. Smears were then treated with methanol for 15 min to permeabilize sperm membranes, followed by incubation for 30 min at room temperature in moisture chambers in phosphate-buffer saline containing 50 p.g/ml FITC-PSA. The smears were then washed three times for 10 min, allowed to dry again, and examined by epifluorescence microscopy (Nikon Microphot-FX, Garden City, NY). Acrosome reactions were diagnosed when a total loss of the acrosomal cap was observed (bar pattern) or no immunofluorescence was seen at all. Duplicate slides were evaluated for each treatment and timeanalyzed, assessed blindly by two experienced observers, and results averaged. At least 200 cells were evaluated per slide. 0.3% HSA) at 37~ in 5% CO 2 in air for 1 h in the presence or absence of H20 2 (Sigma Chemical Co., St. Louis, MO) at different concentrations (0.01 mm through 0.4 ram), and HA and acrosomal status were evaluated. For acrosome reaction studies, the effects of progesterone- 1 ~M-(P4, Sigma) and the calcium ionophore A p~m-(sigma) on HzOz-treated sperm were also examined. Similarly, the effect of different doses of HzO 2 (0.05 mm through 0.2 mm) on sperm-zona pellucida binding was examined in the HZA after 1 h of sperm preincubation (with or without H202) under similar capacitating conditions. Time-course and dose-dependency studies were performed to assess the effect of H202 on [Ca2+]i. These were performed both on previously untreated sperm and following exposure of sperm to P4 (1 txm), the calcium ionophore ionomycin-10 IxM- (Sigma) or catalase-20 ~g/ml-(sigma). Measurement of [Ca2+]i Swim-up sperm (adjusted to 0.5 x 105 motile sperm/ml) were loaded with Fura-2 AM (Molecular Probes, Inc., Eugene, OR) and measurements of [Ca2+]i were performed in a SPEX ARMC spectrofluorometer (Spex Industries, Edison, NJ) following methods described previously (22). Experimental Design The swim-up motile sperm fraction from a given ejaculate was divided into control (medium-treated) and test (H202 or other compound-treated) fractions. Immediately after swim-up, the motile sperm fraction was incubated under capacitating conditions in medium (Ham's F-10 supplemented with Statistical Analysis Analyses of the effect of H20 2 on HA, acrosomal status, and [Ca2+]i (time- and dose-dependency studies) were performed using analysis of variance (ANOVA). HZA results were compared by paired t-tests. Results are expressed as mean +- standard error. P values <0.05 were considered significant. RESULTS The effects of different doses of H202 on HA and acrosome reaction are shown in Table I (n: four ejaculates from four different donors). For HA studies, five doses of H202 were tested from 0.01 through 0.4 ram. Swim-up sperm were incubated Table I. Effect of HzO 2 on HA and Acrosome Reaction a Acrosome reacted sperm (%) Hyperactivated motility b (%) Spontaneous c P4 d A23187 d Control mm mm mM mM mM 0 a Sperm were preincubated in the presence or absence of H202 for 1 h. P4 and A23187 were added after 1 h, and acrosome reaction assessed 30 rain therafter. b p < r P < d Not significant.

4 44 OEHNINGER, BLACKMORE, MAHONY, AND HODGEN Table II. Effect of Various Doses of H202 on Sperm-Zona Pellucida Binding as Tested in the HZA" 200 H202 dose (ram) # sperm bound to hemizona Test b Control b a Sperm were preincubated with H202 for 1 h prior to the HZA. b p < ~ 150 r~ r.. rj / ~ A.control under capacitating conditions for 1 h in the absence (control) or presence (test) of H202. H202 produced a potent and dose-dependent inhibition of HA (ANOVA: F = 20.0, P < ). This inhibitory effect was more pronounced when H202 concentration was/>0.1 mm, with total inhibition of motility at 0.4 mm. Because of this, acrosome reaction and sperm-zona pellucida studies were performed with a maximal concentration of 0.2 mm. H:O2 had a significant effect on the spontaneous rate of acrosome reactions (ANOVA: F = 7.99, P < 0.01). At the concentration of 0.01 mm, H202 produced a marked increase in the percentage of sperm undergoing the spontaneous acrosome reaction as compared with controls. On the other hand, pretreatment with H202 inhibited the stimulatory effect of P4 on the induction of the acrosome reaction but had no effect on A23187-induced acrosome reactions (Table I). HZA results are shown in Table II (three ejaculates from three different donors tested using 10 oocytes per H202 concentration examined). H202 had no effect on sperm-zona pellucida binding at the concentration of 0.05 to 0.1 mm. However, at _ 50 ' ' ' TIME (S~.C) Fig. 2. Time course of the effect of I p~m P4 on [Ca2+] i in the presence or absence of 10 mm H mm H202 significantly inhibited sperm-zona binding (paired t-test, p < 0.004). Figure 1 shows the time-course effects of various concentrations of H202 on basal levels of [Ca:+]/. The three concentrations of H202 tested (0.1, 1.0, and 10 mm) had no significant effects on basal [Ca2+]; over the 40-min time period. The data shown represent the mean --- standard error from three separate experiments using the swim-up fractions of three different donors. Figure 2 depicts the typical time-course effect of 1 IxM P4 on [Ca2+]i using swim-up spermatozoa from a fertile donor in the presence and absence of I0 mm H202. Sperm were loaded with Fura-2 and incubated with or without H202 for 40 min. P4 was added after 10 s of the commencement of data collection. H202 markedly inhibited the initial rapid rise in [Ca2+]i stimulated by P4- Figure 3 presents the time course of various concentrations of H202 to modify the ability of 1 p~m of P4 to elevate r, 140 lz0 = T l x{ ~.~ "~'~ ram H202 "~ ~ o--o 1.0 ram HZ0 e m z~--~ 10.0 mm H202 0 ~ i i i 0 i TIME (rain) Fig. 1. Time course of the effects of various concentrations of HzO2 on basal levels of [Ca:+]i. z~--zx lo.o ~ ~2o2 0 i i f I TIME (min) Fig. 3. Time course of the effect of various concentrations of H202 to modify the ability of 1 txm P4 to elevate [Ca2+] i.

5 DUAL EFFECTS OF H202 ON HUMAN SPERM FUNCTION 45 [Ca2+] i. Sperm were treated with either 0.1, 1.0, or 10 ~ H202. After various times (0, 10, 20, and 40 min) the ability of 1 p~m P4 to increase [Ca2+] i was measured. Both 1.0 and 10 mm H202 significantly inhibited (P < 0.05) the ability of P4 to increase [Ca2+]i in a time-dependent manner; 0.1 mm H202 had minimal effect. These data represent the mean -+ standard error from three separate experiments using the swim-up fractions of three different donors. Figure 4 shows the time course of the effect of 1 p.m P4 on [Ca2+]i in sperm that were treated for 25 rain with catalase (20 ixg/ml), H202 (10 ram), or not treated (control). The effect of P4 to initially increase [Ca2+]i was blunted by H202 and potentiated by catalase. These data represent the average results from two different experiments using the swim-up fractions of two different donors. The dose-response of H2Oz on the ability of the calcium ionophore ionomycin to increase [Ca 2+ ]i in swim-up sperm is shown in Fig. 5. H202 was added to Fura-2 loaded sperm at the indicated concentrations for 50 rain. Ionomycin (10 p.m) was added, and the maximum elevation of [Ca 2+ ]i was measured for each H202 concentration. As depicted, the ability of ionomycin to increase [Ca 2 + ]i was slightly potentiated (although not significantly different) by 1.0 and 10 mj~ H202 treatment. The data shown represent the mean +_ standard error of three separate experiments using swim-up sperm from three different donors. DISCUSSION At high concentrations H202 produced a deleterious effect on sperm functions crucial to fertiliza- 500 ~ 400 r..3 ~ 300 ~ 2oo rj z too TIME (SEC) Fig. 4. Time course of the effect of 1 ~M P4 on [Ca2+]i in sperm treated with catalase, HzOz, or untreated. "~ 160 [ ~J o ~.~ 14o I 1 ~ ~ lo0 ~ N 80 b 50 rain treatment with H202./ ' II ' ' ' C 0.i 1.0 I0.0 H202 mm Fig. 5. Dose response of H2O 2 on the ability of 10 IxM ionomycin to increase [Ca2+]i in sperm. tion. This was maximally observed with a concentration/>0.4 mm which totally inhibited all motility parameters (determining a complete abolition of HA), but significant negative effects were already seen at 0.1 mm. This was associated with a diminished sperm capacity to achieve tight binding to the zona pellucida in the HZA. We postulate that a decrease in sperm motion parameters, including the ability to demonstrate HA, were causative factors leading to an impaired sperm-zona pellucida interaction. H202 appears to be the most toxic ROS toward human spermatozoa, and this has been demonstrated as irreversible motility arrest probably due to membrane lipid peroxidation. There is evidence to suggest an association between decreased capacity for sperm-oocyte fusion (using the hamster zonafree egg system) and detectable ROS levels (23). Furthermore, it has recently been shown that the presence of leukocytes in sperm preparations of infertile men was associated with elevated levels of ROS production, impaired movement, and a reduced capacity for fertilization in vitro (24). In the absence of leukocytes, generation of ROS by spermatozoa was correlated with loss of sperm motility (24). Our studies confirm and extend those observations by showing a dose-dependent effect of H202 on HA and on sperm-zona pellucida binding capacity using a homologous, internally controlled bioassay. It has been reported that H202 directly affected the rate of sperm-oocyte fusion in the hamster zonafree egg system at a concentration of 0.2 mm (25). In those studies, H202 did not affect motility but decreased the percentage of sperm undergoing the acrosome reaction. Those studies were carried out

6 46 OEHNINGER~ BLACKMORE~ MAHONY~ AND HODGEN after exposing sperm cells to the xanthine oxidase system and the calcium ionophore A Here, under different experimental conditions (capacitating conditions in culture medium) we observed loss of HA at levels/>0.1 mm. Since this determined a decreased number of motile spermatozoa exposed to the zona pellucida, the impaired HZA results are probably due to a diminished number of motile cells in the incubation droplet. Whether other defects were also causative for a lower capacity to bind to the zona pellucida (i.e., membrane alterations) remains to be clarified. Higher concentrations of H202 (>/1.0 mm) interfered with the capacity of sperm to respond to a P4 challenge with an increase in [Ca2+]i (although H202 itself did not directly interfere with the basal intracellular concentration of calcium). Conversely, catalase potentiated the stimulatory effect of P4. This suggests that membrane/ intracellular alterations also may be responsible for a decreased functional ability to interact with the zona pellucida and the oocyte itself in the presence of high levels of H202. Additionally, the ability of ionomycin to increase [Ca2+]i was slightly potentiated by 1.0 and 10 mm H202 treatment. This indicates that H202 is not preventing Fura 2 from being able to measure [Ca2+]i in the spermatozoa. We speculate that the potentiation of [Ca2+] i may be related to H202 inhibiting the plasma membrane CaE---ATPase pump. It has been shown that the superoxide anion (generated by a combination of xanthine plus xanthine oxidase in the presence of catalase) triggered higher levels of HA and capacitation of human spermatozoa than did fetal cord serum, a known biological inducer of these processes. The presence of superoxide dismutase (the enzyme responsible for the degradation of the superoxide anion to hydrogen peroxide) in the incubation medium blocked the induction of spontaneous and superoxide anioninduced HA and capacitation (14). The fact that addition of superoxide dismutase to already hyperactivated spermatozoa reversed the HA suggested that sperm needed sustained superoxide generation to maintain HA and complete capacitation. Moreover, studies in hamster sperm suggested that H202 produced by sperm (14,26) cells could play a significant role during the process of capacitation. For these reasons, we investigated whether low concentrations of H202 could have an effect opposite that of high concentrations (i.e., stimulation of HA and acrosome reaction under capacitating conditions). Our results showed that low concentrations of H202 (0.01 to 0.1 mm) were able to maintain HA and sperm-zona pellucida binding capacity and directly enhanced the acrosome reaction. Furthermore, at these low concentrations H202 did not affect the ability of A23187 to induce the acrosome reaction, although it blunted Pn-induced acrosome reaction. This suggests that although H202 can directly maintain HA and spontaneous acrosome reaction, even low levels of this ROS may produce detrimental changes, as evidenced by an attenuation of Pa-mediated events (by interfering with membrane/signal transduction/intracellular mechanisms) (27,28). We conclude that H202 directly affects sperm functions crucial to fertilization in a dual dose- and time-dependent fashion in human spermatozoa. Low concentrations maintain HA and the ability to undergo spontaneous acrosome reaction and achieve tight binding to the zona pellucida, whereas higher concentrations have significant deleterious effects as determined by the end points of the capacitation process. These effects seem to be dependent on modifications of plasma membrane and intracellular homeostasis by the oxidative process. ACKNOWLEDGMENTS We thank Ms. Dara Willett-Leary for her editorial assistance. REFERENCES 1. Gagnon C, Iwasaki A, de Lamirande E, Kovalski N: Reactive oxygen species and human spermatozoa. In The Male Germ Cell, B Robaire (ed). Ann NY Acad Sci 1991 ;637: Aitken RJ, Harkiss D, Buckingham DW: Analysis of lipid peroxidation mechanisms in human spermatozoa. Mol Reprod Dev 1993;35: Jones R, Mann T, Sherins RJ. Peroxidative breakdown of phospholipids in human spermatozoa, spermicidal properties of fatty acid peroxides and protective action of seminal plasma. Fertil Steril 1979;31: Aitken RJ, West KM: Analysis of the relationship between reactive oxygen species production and leukocyte infiltration in fractions of human semen separated on Percoll gradients. Int J Androl 1990; 13: Alvarez JG, Touchstone JC, Blasco L, Storey B: Spontaneous lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. Superoxide dismutase as a major protectant against oxygen toxicity. J Androl 1987;8: Aitken JR, Clarkson JS: Cellular basis of defective sperm

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